A novel approach to control Botrytis cinerea fungal infections: uptake and biological activity of antifungals encapsulated in nanoparticle based vectors

Botrytis cinerea, responsible for grey mold diseases, is a pathogen with a broad host range, affecting many important agricultural crops, in pre and post harvesting of fruits and vegetables. Commercial fungicides used to control this pathogen are often subjected to photolysis, volatilization, degradation, leaching, and runoff during application. In this context, the use of a delivery system, based on poly (lactic-co-glycolic acid) nanoparticles (PLGA NPs) represents an innovative approach to develop new pesticide formulations to successfully fight B. cinerea infections. In order to study NPs uptake, B. cinerea conidia and mycelium were treated with PLGA NPs loaded with the high fluorescent probe coumarin 6 (Cu6-PLGA NPs) and analyzed under ApoTome fluorescence microscopy. The observations revealed that 50 nm Cu6-PLGA NPs penetrated into B. cinerea conidia and hyphae, as early as 10 min after administration. Pterostilbene, a natural compound, and fluopyram, a synthetic antifungal, were entrapped in PLGA NPs, added to B. cinerea conidia and mycelium, and their antifungal activity was tested. The results revealed that the compounds loaded in NPs exhibited a higher activity against B. cinerea. These results lay the foundations for the use of PLGA NPs as a new strategy in plant pest management.

Fungal plant pathogens are the main cause of considerable economic losses of crop plants 1 . Ascomycete Botrytis cinerea, responsible for grey mold diseases, is a highly successful pathogen due to its flexible infection modes, high reproductive output, wide host range, and ability to survive for extended periods as conidia and/or small hardened mycelia masses called sclerotia 2 . It is a pathogen with a broad host range, affecting many important agricultural crops, mainly in pre and post harvesting of fruit or vegetables. It is estimated that B. cinerea causes a $10 to 100 billion of production loss annually worldwide 3 . Due to the highly destructive nature of B. cinerea, it was ranked second on a list of fungal pathogens of scientific and economic importance 4 . Approximately 8% of the global fungicide market is used to control this pathogen. It is important to highlight that less than 0.1% of fungicides reach their biological targets as 90% of them are lost through photolysis, volatilization, degradation, leaching, and runoff during application 5 . In addition, fungicide usage is harmful to both the environment and human health, and B. cinerea has developed resistance to many conventional fungicides such as dicarboximides and benzimidazoles 6 .
To overcome the fungal multi-resistance to existent drugs, it is important to explore novel antifungal agents, which may replace current control strategies. Some authors reported the activity of plant extracts or natural compounds against B. cinerea 7 . In this context, the generation of drug delivery systems based on nanomaterials represents a potential alternative to develop novel formulations to successfully combat fungal infections and overcome the fungal multi-resistance to existent drugs 8  www.nature.com/scientificreports/ Different studies have determined that NPs present fewer side effects and greater specificity to the infection site 9,10 . Special attention has been given to biopolymeric nanoparticles (NPs) as non-toxic and eco-friendly nanocarriers that can be successfully used for the controlled release of bioactive compounds 11 . In recent years, a variety of natural and synthetic polymers have been explored for nanoformulations. Among them, polylactic acid (PLA), polyglycolic acid (PGA), and their copolymer (PLGA) have been extensively investigated due to their biocompatibility, high solubility, stability, and effectiveness [12][13][14][15] .
It has been shown that PLGA NPs have the ability to penetrate Vitis vinifera cell suspensions without harmful effects on cellular vitality 16,17 . As largely investigated by the authors in V. vinifera cell cultures, methyl jasmonate (MeJA) encapsulation in PLGA NPs significantly promoted MeJA cell uptake and the activation of MeJA-induced responses 13 . Simonetti et al. 15 have demonstrated the anti-Candida biofilm activity of PLGA NPs loaded with non-fermented grape pomace.
Up to now, there is relatively little and limited evidence about PLGA NPs uptake by pathogenic fungal cells 18,19 , and further studies are needed in order to improve the knowledge on the effectiveness of PLGA NPs to deliver natural or conventional antifungals to microbial cells.
The present research aimed to study PLGA NPs uptake in B. cinerea and investigate the antifungal activity of natural and synthetic compounds entrapped in PLGA NPs against B. cinerea conidia and mycelium.
In order to examine NPs uptake, B. cinerea conidia and mycelium were treated with PLGA NPs loaded with the high fluorescent probe coumarin 6 (Cu6-PLGA NPs) and analyzed under ApoTome fluorescence microscopy. Moreover, in an effort to investigate the antimicrobial activity of antifungals delivered by NPs, pterostilbene and fluopyram were encapsulated in PLGA NPs and administered at different stages of B. cinerea development.
Pterostilbene is a natural antimicrobial phenolic compound derived from resveratrol, mostly contained in V. vinifera leaves and grape berries. It has been demonstrated that pterostilbene has no harmful effects on plant metabolism or crop yield, and it is able to inhibit the growth of several phytopathogenic fungi, such as Leptosphaeria maculans, Peronophythora litchii, Botrytis cinerea and others 20,21 . Schmidlin et al. 22 showed that pterostilbene was 5 to 10 times more effective than resveratrol in inhibiting the germination of conidia of B. cinerea and sporangia of P. viticola. On the other hand, fluopyram is a synthetic fungicide and nematicide compound commonly used in agriculture against B. cinerea.
This study provides new evidence of the use of PLGA NPs as an interesting strategy in integrated plant disease management, with the aim to increase the potency and efficiency of natural and conventional antifungals through a controlled and targeted drug release while decreasing environmental toxicity and agricultural costs.

Materials and methods
Chemicals and materials. Pterostilbene was purchased from Chemodex (St. Gallen, Switzerland).
Potato dextrose broth (PDB) was purchased from Formedium LTD (Hunstanton, Norfolk, England). The microfluidic flow focusing reactor was assembled by the research group involved in the study as reported previously by Bramosanti et al. 23 .
Synthesis of PLGA NPs. PLGA NPs loaded with fluopyram, pterostilbene or coumarin 6 were prepared by using an innovative microfluidic reactor with a flow-focusing configuration described previously 24 . The reactor consists of three inlets and one main mixing outlet channel. An organic phase containing the polymer is injected into the middle channel and water is injected into the two side inlets. NPs formation occurs through a nanoprecipitation mechanism in the mixing channel and NPs can be recovered at its end. PLGA (2 mg mL −1 ) and the selected payload were dissolved in an organic phase. Acetone was used to dissolve pterostilbene (1 mg mL −1 ) and coumarin 6 (40 μg mL −1 ), while fluopyram (0.2 mg mL −1 ) was dissolved in DMSO. To optimize the amount of encapsulated fluopyram and pterostilbene, PLGA NPs were prepared with different polymer/drug ratios, respectively from 2.5:1 to 20:1 and from 2:1 to 4:1, keeping PLGA concentration constant. A PLGA/coumarin 6 ratio of 50:1 was chosen on the basis of previous works 24 . The aqueous flow rate was 2000 µL min −1 while the organic phase flow rate was 100 μL min −1 when using acetone and 400 µL min −1 when using DMSO. The formed PLGAbased NPs were recovered and the organic phase was eliminated under reduced pressure. The NPs aqueous suspensions were stored at 4 °C until use.

PLGA NPs characterization.
Dynamic light scattering (DLS) measurements were carried out using a NanoZetasizer (Malvern Instruments, Malvern, UK) to measure the mean hydrodynamic diameter of PLGA NPs and their polydispersity index. The experimental conditions used are the following: a helium neon laser operating at 633 nm, a fixed scattering angle of 173° and constant temperature (25 °C). The measured autocorrelation functions of the scattered light intensity were analyzed using the CONTIN algorithm in order to obtain the decay time distributions 25 . Decay times were used to determine the distributions of the diffusion coefficients of the particles (D), converted in turn in the distributions of the apparent hydrodynamic radii, R H , using the Stokes-Einstein relationship: R H = k B T/6πηD (kBT = thermal energy; η = solvent viscosity).
Particle morphology was observed by scanning electron microscopy (SEM) in both the secondary and the backscattered electron modes with an electron acceleration voltage of 20 keV, using a LEO 1450VP SEM microscope (ZEISS, Oberkochen, Germany).
The quantitative analysis of fluopyram and pterostilbene loaded in PLGA NPs was carried out by spectroscopic measurements. NPs aqueous suspensions were ultra-centrifuged at low temperature (4 °C) to recover NPs. The Scientific Reports | (2022) 12:7989 | https://doi.org/10.1038/s41598-022-11533-w www.nature.com/scientificreports/ supernatant was discarded and the pellet was dissolved in DMSO and analyzed by measuring the UV absorbance at 270 nm (for fluopyram) or at 313 nm (for pterostilbene), comparing the results with the corresponding calibration curve. The method used for fluopyram was linear within the concentration range between 0.2 and 0.8 mg mL −1 with R 2 = 0.997. The method used for pterostilbene was linear within the concentration range between 0.002 and 0.01 mg mL −1 with R 2 = 0.9822. The encapsulation efficiency and loading capacity were calculated by using the following equations: In vitro release studies of fluopyram and pterostilbene from PLGA NPs. A fixed amount (2 mg Fluorescent analysis. Cu6-PLGA NPs treated conidia and mycelium were observed and images were acquired using an Axio Imager M2 fluorescence microscope (Zeiss, Germany), motorized on the 3 axes, using a FITC filter (λ excitation BP 455-495 nm; λ emission BP 05-555 nm). The high thickness of the sample required a Z-stack image scanning performed with an Axiocam 512 (Zeiss) monochromatic camera and ApoTome 2 (Zeiss) as fringe projection module used to remove the out of focus signal. Single plane images were obtained as Z-stack maximum projection using Zen 2.5 (Zeiss) image analysis software.
In vitro antifungal activity of pterostilbene and fluopyram. In vitro antifungal activity was carried out as previously described by Meletiadis et al. 28 Table 1. According to the used experimental conditions, PLGA NPs entrapping fluopyram, pterostilbene, or coumarin 6, presented an average size of 50 nm, with polydispersity indexes below 0.2. Investigations by scanning electron microscopy (SEM) showed that drug-loaded PLGA NPs had a spherical morphology (Fig. 1). Drug loading efficiencies and loading capacities for fluopyram-PLGA NPs or pterostilbene-PLGA NPs prepared with different polymer/drug ratios are reported in Tables 2 and 3 respectively. The PLGA/fluopyram and PLGA/pterostilbene weight ratios that showed the best encapsulation efficiency and loading capacity were respectively 10:1 and 2:1. These conditions were selected for all successive observations. In vitro release kinetics of fluopyram and pterostilbene from PLGA NPs. The in vitro release of fluopyram occurs slowly reaching a plateau after 120 h as a function of medium pH (Fig. 2a). In both experimen-    . Nevertheless, the release of the drug appears to be positively influenced by lower pH; in these experimental conditions, acid hydrolysis of the polymeric backbone could be favoured, which would influence the release of the drug by erosion. The amount of pterostilbene released within the time interval examined was low, probably due to the poor solubility of pterostilbene in water (0.011 g L −1 ) and the hydrophobic interactions that stabilize the PLGA-pterostilbene complex. No significant change in the amount of released pterostilbene as a function of pH was observed (Fig. 2b). The % of pterostilbene released at pH 4, at the plateau conditions, was equal to 16% and corresponded to 30 μg mL −1 , an amount compatible with the in vitro inhibition protocols of B. cinerea, in which pterostilbene was used at different concentrations between 20 and 1.25 μg mL −1 . However, such results could be useful to compare in vitro and in vivo performances since in in vivo experiments other mechanisms that favor NPs erosion phenomena may occur.

Microscopic observations of NPs uptake in B. cinerea conidia and mycelium.
According to the microscopic analyses, Cu6-PLGA NPs administered to B. cinerea enter conidia and hyphae after 10 min. It was found that NPs interacted with the conidia wall from the first stages of germination before the formation of the septum (Fig. 3a,b). The conidia of Botrytis cinerea have relatively thick cell walls (0.5 μm) consisting of two layers, a thin, dark, rough outer layer that is electron-dense under the microscope, and a thicker inner one that is transparent under electron microscopy 29 . Based on fluorescence results, optical sections and the 3D reconstruction of the ApoTome Microscope, it has been possible to observe that the fluorescent signal was present both along the conidia wall and inside conidia, throughout the whole thickness of the spore (Fig. 3c). The NPs fluorescent signal was well visible also in the expanding germ tube (Fig. 3d,e). As the tube elongates, a large void forms in the center of the original spore.
The 50 nm Cu6-PLGA NPs were also administered to B. cinerea mycelium. 1 h after NPs administration, an intense signal appeared in the fungal hyphae ( Fig. 3f-h). Fluorescence was visible in the mycelium after washing with sterile saltwater, showing that NPs penetrated or remained adhered to the hyphae. No autofluorescence was detected from the conidia and mycelium alone (data not shown). In a previous work, we demonstrated that PLGA NPs were able to penetrate the mycelium of some plant pathogenic fungi such as A. carbonarius, A. niger and B. cinerea 16 . During B. cinerea conidia germination, the outer spore wall breaks, and the emergent germ tube appears surrounded by the elastic inner one and by a mucilage sheath. At a very early stage, a transverse wall with a central pore is laid at the base of the germ tube 29 . Similarly, the uptake of Cu6-PLGA NPs has been previously demonstrated in fungal cells of Aspergillus flavus. In particular, Patel et al. 12 showed that the uptake of Cu6-PLGA NPs in A. flavus spores and mycelium was closely related to NPs size. In such work, NPs of 203 nm interacted with fungal cell surfaces and were efficiently internalized after 1 h of incubation. Muse et al. 30 observed that covalently tagged poly(lactic-co-glycolic) acid nanoparticles (PLGA-tetramethylrhodamine [PLGA-TRITC]), entrapping coumarin 6 (double-tagged) with a diameter of 85-150 nm released coumarin 6 in A. flavus spores and hyphae while the majority of the particles themselves did not seem to be trafficked into the interior of the cells. Nonetheless, some red fluorescence (PLGA-TRITC) was still observed within the interior of the cell, indicating that the smaller nanoparticles (30-50 nm) may have been directly endocytosed or diffused through the hyphae cell wall 30 . Other studies were carried out to test biopolymeric drug delivery systems against B. cinerea. In a recent work, Raj and collaborators 31 showed that chitosan-arabic gum-coated liposome 5I-1H-indole nanoparticles were potent inhibitors against B. cinerea growth. Up to now, despite B. cinerea causing significant damage to agriculture every year, few data are available on its uptake of PLGA NPs. The inhibition of spore germination should be considered effective for controlling plant disease. In this work, we showed that PLGA NPs interacted and penetrated also in the conidia of B. cinerea, promoting the uptake of the encapsulated compound into fungal cells, with the aim to prevent spore germination and thus fungal infection.  www.nature.com/scientificreports/ the free compounds (Fig. 4). In both experiments, empty NPs did not show antifungal activity. NPs loaded with pterostilbene, added to germinated conidia, showed significant inhibition of B. cinerea growth after 24 h of incubation. NPs loaded with fluopyram significantly inhibited B. cinerea growth, compared to free fluopyram, after 72 h of incubation, in all tested concentrations (Fig. 5). The antifungal activity of pterostilbene, a well-known plant phytoalexin, on B. cinerea has already been demonstrated by Pezet and Pont since 1990 32 . More recently, Xu and collaborators 21 reported 45% of mycelial growth reduction after treatment of B. cinerea with 200 µg mL −1 of pterostilbene. Moreover, Xu and collaborators 33 in a subsequent study showed multiple action mechanisms of pterostilbene against B. cinerea. After treatment with pterostilbene, B. cinerea changed the morphology of the hyphae and conidiophores, lost plasma membrane integrity, modulated gene expression, and increased laccase production. Some researchers reported that B. cinerea, through laccase secretion, can degrade and neutralize the toxicity of pterostilbene 34 . Our results show a significant and long lasting increase in activity, even at low concentrations. The significant increase in activity of pterostilbene delivered by PLGA NPs could be due to the protection of pterostilbene from fungal laccase. Moreover, the higher hydrophobicity of pterostilbene delivered by PLGA NPs could increase diffusion through fungal membranes. On the other hand, pterostilbene with mul-  www.nature.com/scientificreports/ tiple mechanisms of action carries a low risk of developing resistance and its low toxicity makes pterostilbene a winning compound to fight B. cinerea. Synthetic fungicides are currently widely used in agriculture to fight B. cinerea infections. In this context, fluopyram, a synthetic pyridinyl ethylbenzaimide fungicide that inhibits succinate dehydrogenase (SDH), is commonly used to safeguard crops. Some studies reported high risks of B. cinerea resistance development to fluopyram and other succinate dehydrogenase inhibitors 35 . However, Dong and Hu 36 have shown that the transformation products were more toxic than fluopyram. Moreover, fluopyram shows high residue in surface soil which affects subsequent crops 37 . In this study, NPs loaded with fluopyram significantly inhibited B. cinerea growth, compared to free fluopyram. Agriculture is the backbone of the economy of most countries and it was the key development in the rise of human civilization. B. cinerea is a well-known fungus with a wide host range that causes heavy losses in crop yields every year. The generation of drug delivery systems based on nanomaterials represents a potential alternative to developing newer formulations to successfully control fungal infections and overcome the fungal multiresistance to existent drugs. In fact, the encapsulation protects the antifungal compound from damage such as photolysis or degradation, allowing the drug to reach the target site more efficiently with a consequent reduction in the number of applications 38,39 . In this context, the delivery of antifungal compounds encapsulated in PLGA NPs could be considered an excellent strategy to safeguard crops against B. cinerea. The fluorescence microscopy observations revealed that 50 nm Cu6-PLGA NPs penetrated B. cinerea conidia and hyphae as early as 10 min after administration. Moreover, the antifungals pterostilbene and fluopyram delivered by NPs exhibited higher antifungal activity against B. cinerea than the corresponding antifungal compounds administered in free form. These results lay the foundation for the use of PLGA NPs in agriculture as a new strategy in plant pest management with the goal of enhancing the effectiveness of natural and synthetic antifungals through controlled and targeted drug delivery in the agronomic field.